The general goal of this grant proposal is to define a signal transduction pathway for tumor necrosis factor (TNF)-alpha. This cytokine is a principal host defense against foreign antigen and may serve a variety of physiologic regulatory functions. Although a number of reports have suggested a role for one of the known signalling systems, no coherent picture has emerged for a second messenger pathway that may account for the entirety of the pleiotropic effects of TNF-alpha. This proposal is based on the recent observation that sphingomyelin degradation to ceramide is an early event in TNF-alpha action in human promyelocytic leukemia (HL-60) cells and that ceramide may substitute for TNF-alpha in monocytic differentiation. During the past few years, this laboratory has described a new metabolic pathway involving sphingomyelin and its derivatives. This pathway is initiated by hydrolysis of plasma membrane sphingomyelin to ceramide by the action of a sphingomyelinase. Subsequently, ceramide may be de-acylated to sphingosine, an inhibitor of protein kinase C, or phosphorylated to ceramide 1-phosphate, a compound newly discovered in this laboratory. Because of apparent similarities between this metabolic pathway and the phosphoinositide pathway, it was postulated that ceramide may serve second messenger function. Indeed, a cell-permeable ceramide analog induced selective phosphorylation of the epidermal growth factor receptor on threonine 669. Based on this observation, this laboratory has recently characterized a novel kinase that mediates this action of ceramide, termed ceramide-activated protein kinase, in membranes derived from HL-60 and A431 human epidermoid carcinoma cells. Elevation of ceramide levels by TNF-alpha also resulted in enhanced kinase activity in membranes derived from stimulated cells. The specific aims of this grant are to: (1) demonstrate that this sphingomyelin pathway is tightly coupled to activation of the TNF receptor both in intact cells and in vitro: (2) show that this pathway is sufficient to mediate TNF-alpha-induced protein phosphorylation; and (3) generalize this pathway to various models of TNF- alpha action and in particular determine how it might relate to TNF-induced cytotoxicity and cytostasis. Hopefully, these studies will provide fundamental insights into the mechanism of TNF-alpha action and allow for eventual pharmacologic manipulation of this system.